The present invention relates to a sulfatase and a method for its production by fermentation. More particularly, it relates to a sulfatase isolated from a species of Streptomyces.
One of the major problems in human cancer chemotherapy is the nonspecific action of antitumor agents which can cause unwanted damage to normal cells. Numerous attempts have been made to more selectively deliver a cytotoxic agent to or near the tumor site thereby minimizing toxicity to normal tissues. A great deal of effort in this area has been devoted to linking a cytotoxic agent to a second component which may have a higher affinity for tumor cells than for normal cells, for example an antibody, a hormone, a lectin, or a polymer.
More recently, a different approach has been proposed which involves administering to a tumor bearing host a prodrug of an antitumor agent in conjunction with an antibody-enzyme (ab-enz) conjugate [see, e.g., P. D. Senter, et al., European Application 302,473, published Feb. 8, 1989]. The conjugate consists of an enzyme that is capable of converting the prodrug into the active parent compound and a tumor-specific antibody which serves to bring the enzyme to the tumor cell surface where the enzyme would act on the prodrug. This method can, thus, potentially create a higher concentration of the antitumor drug in the vicinity of the tumor to which the ab-enz conjugate is bound. For use in the ab-enz conjugate/prodrug approach, the enzyme is preferably one that is not present in the blood stream in very high concentration in order that the prodrug may remain intact until it encounters the enzyme at the tumor site. The prodrug itself may be considerably less cytotoxic than the parent drug; the cytotoxic drug may be one of the commonly used antitumor agents that is amenable to modification to produce a prodrug which can regenerate the parent drug enzymatically.
Etoposide (Ia) and teniposide (Ib) are two clinically established antitumor drugs belonging to a class of compounds generally known as 4'-demethylepipodophyllotoxin glucosides (DEPG). The general structure of 4'-demethylepipodophyllotoxin glucosides is depicted below as formula (I) wherein R.sup.1 may be, for example, C.sub.1-10 alkyl, 2-thienyl, furyl, or phenyl: ##STR1## The hydroxyl groups and phenol group of DEPG may be derivatized to provide a suitable prodrug as substrate for an ab-enz conjugate. In fact, the effectiveness of etoposide 4'-phosphate in combination with a monoclonal antibody-alkaline phosphatase conjugate has been demonstrated in a murine human colon carcinoma xenograft model [Senter, et al., supra].
In addition to etoposide 4'-phosphate, etoposide sulfates are also compounds known in the art. These derivatives are disclosed in Japanese Kokai 88/192,793 and are depicted as formula (II) below: ##STR2## wherein one of A, B, and X is the group --SO.sub.3 H and the others are H.
Etoposide 4'-sulfate (A=B=H; X=--SO.sub.3 H) appears to be much less cytotoxic than etoposide itself, requiring a very large dose to achieve the same degree of activity as etoposide. This may indicate that etoposide 4'-sulfate is not facilely converted into etoposide in vivo and, thus, may be more suitably used as a prodrug in combination with an ab-enz conjugate. The enzyme needed to effect the conversion of etoposide 4'-sulfate into etoposide would be a sulfatase which catalyzes the hydrolysis of a sulfate ester to the corresponding hydroxyl compound as follows: ##STR3##
Several types of sulfatase have so far been studied, and these include Type I and Type II arylsulfatases, steroid sulfatases, glycosulfatases, choline sulfatases, alkylsulfatases, and myrosulfatases. (For a review on sulfatases, see "The Hydrolysis of Sulfate Esters" by A. B. Roy in "The Enzymes", vol. V, pp. 1-19, P. D. Boyer, Ed., Academic Press, 1971.) Among these, arylsulfatases (aryl sulfate sulfohydrolase, EC 3.1.6.1), which catalyzes the above reaction where R is an aromatic ring, have been isolated from various animal tissues, as well as microbial sources, and have been most extensively studied (for a review on arylsulfatases, see "Arylsulfatases" by R. G. Nicholls and A. B. Roy, ibid, pp. 21-41). It is noteworthy that, even though many sulfatases have been reported, few have been purified to homogeneity. For example, arylsulfatase A from rabbit liver has a molecular weight of approximately 70 kD (monomer), forms a dimer at pH 7.4 and tetramer at pH 4.8, and has been purified 10,000 fold (G. D. Lee and R. L. Van Etten; Arch. Biochem. Biophys., 166, 280-294, 1975); arylsulfatase isolated from ox liver is a glycoprotein having a molecular weight of 107 kD (monomer) (L. W. Nichol and A. B. Roy; J. Biochem., 55, 643-651, 1964 and E. R. B. Graham and A. B. Roy; Biochim. Biophys. Acta, 329, 88-92, 1973).
Arylsulfatase activity in releasing sulfate from lignosulfonate was demonstrated in cell free extracts of Streptomyces sp. L.; however, the enzyme itself was not purified or characterized (Y. L. Steinitz, Eur. J. Appl. Microb. Biotechnol., 13, 216-221, 1981).
Arylsulfatases isolated from various sources are available commercially. These enzymes have been evaluated using etoposide 4'-sulfate as the substrate; however, most of these showed either no or little hydrolytic activity against this compound. Furthermore, none of the commercially available sulfatases are homogeneous and some have unfavorable characteristics such as high molecular weight, low optimum pH, etc., rendering them unsuitable for clinical use.
Against this background, a program was initiated to screen for microbial arylsulfatases which are capable of hydrolyzing a 4'-demethylepipodophyllotoxin glucoside 4'-sulfate to the corresponding 4'-demethylepipodophyllotoxin glucoside. The result of this effort forms the basis of the present invention.